LpxR and PagL expression in live attenuated auxotrophic Pseudomonas aeruginosa vaccines modulates lipid A reactogenicity in vitro while preserving immunogenicity

Miriam Moscoso^1,2Víctor Fuentes-Valverde^2,3,4Patricia Garcia Fernandez^2,3Juan Andrés Vallejo³Jesús Pérez-Ortega⁵Rebeca Santamarina-Fernández³Ana Candela³Marina Oviaño^2,3Jesús Arenas^5,6*German Bou^2,3,7*

• ¹Complexo Hospitalario Universitario de A Coruña; Servicio de Microbiología; Instituto de Investigación Biomédica de A Coruña; Servizo Galego de Saúde, A Coruña, Spain
• ²Centro de Investigacion Biomedica en Red Enfermedades Infecciosas, Madrid, Spain
• ³Complexo Hospitalario Universitario de A Coruña, Servicio de Microbiología; Instituto de Investigación Biomédica de A Coruña; Servizo Galego de Saúde, A Coruña, Spain
• ⁴Agencia Española de Medicamentos y Productos Sanitarios, Área de Medicamentos Biológicos, Madrid, Spain
• ⁵Utrecht University, Faculty of Science and Institute of Biomembranes, Department of Molecular Microbiology, Utrecht, Netherlands
• ⁶University of Zaragoza, Faculty of Veterinary, Unit of Microbiology and Immunology; Agroalimentary Institute of Aragon, Zaragoza, Spain
• ⁷Universidad de A Coruña, Departamento de Fisioterapia, Medicina y Ciencias Biomédicas, A Coruña, Spain

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen responsible for severe respiratory tract infections. We previously developed a live attenuated auxotrophic vaccine candidate, PAO1 ΔmurI, which conferred protection in murine infection models but exhibited significant reactogenicity when administered intranasally. To reduce the toxicity of PAO1 ΔmurI without compromising its protective efficacy, we engineered strains with a modified lipid A structure, as lipid A is one of the main toxic components of whole-cell vaccines. Specifically, we overproduced two lipid A-modifying enzymes: LpxR and PagL. Purified lipopolysaccharide (LPS) was analyzed by MALDI-TOF mass spectrometry. LPS extracted from the wild-type strain showed heterogeneous lipid A structures with varying degrees of acylation, but a predominant penta-acylated species. Expression of LpxR led to enrichment in tetra-acylated species, while PagL expression reduced the heterogeneity observed in the wild type. In vitro assays using HEK293-Blue reporter cells expressing murine and human Toll-like receptor 4 (TLR4) showed decreased TLR4 activation by both mutant strains. In a murine model of acute pneumonia, both lipid A-modified derivatives retained protective efficacy; however, in contrast to in vitro findings, no reduction in reactogenicity was observed in vivo. Our results suggest that while lipid A modifications mediated by LpxR and PagL can attenuate TLR4 signaling, additional modifications or targeting of other toxic components may be required to reduce in vivo reactogenicity of our vaccine. This strategy may serve as an initial basis for optimizing live attenuated P. aeruginosa vaccines, although additional approaches will likely be necessary to achieve substantial improvements.